Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation

Abstract

Targeted human cytolytic fusion proteins (hCFPs) are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv) specifically binding to e.g., tumor associated antigens (TAAs). After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to up-regulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively), reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD) simulations and enhanced sampling methods (ESM). MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such “high-resolution” detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields.

Critical Issues in the Development of Immunotoxins for Anticancer Therapy

“…18 Finally, recent fourth-generation ITs consist of human or humanized antibody fragments fused to human-derived cytotoxic proteins, such as cytotoxic enzymes (proteases and RNases) and cytostatic proteins, to minimize the off-target toxicity and immunogenicity. 8,21,22 In addition to the aforementioned development-based categorization, 4,9 ITs can be further classified according to (1) the targeting moiety, (2) toxin moiety, and (3) targeting diseases. First, ITs according to the type of targeting moiety are divided into 3 groups including ligand-, antibody-, and peptide-based ITs.…”

“…92 To prevent the hypersensitivity and intrinsic immunogenicity associated with nonhuman toxins, human endogenous cytotoxic proteins have been utilized as an alternative payload in fourth-generation ITs. 21,93 Such ITs have been generated based on GrB, 94,95 angiogenin, 96 pancreatic RNase A, 8 microtubule-associated protein tau, 97 and deathassociated protein kinase 98 and have shown in vitro and in vivo antitumor activities. Nonetheless, the cytotoxicity of human enzymatic toxins is compromised by endogenous inhibitors in the cytosol: GrB is suppressed by serine protease inhibitor B9 (serpin B9 or PI9), 22 and pancreatic RNase A by the ribonuclease inhibitor (RI) protein.…”

“…Nonetheless, the cytotoxicity of human enzymatic toxins is compromised by endogenous inhibitors in the cytosol: GrB is suppressed by serine protease inhibitor B9 (serpin B9 or PI9), 22 and pancreatic RNase A by the ribonuclease inhibitor (RI) protein. 8,21 For instance, RI binds to RNase A with high affinity (fM range) protecting the cell from an RNase attack. 99 Accordingly, the variants of RNase A 100 and GrB 101,102 that are resistant to the binding of a natural inhibitor have been engineered to maximize the cytotoxic effects.…”

“…Nonetheless, the in vivo antitumor activity of the human enzyme variantebased ITs is much lower than that of PE38-and DT-based ITs. 21,61,93 Furthermore, human cytotoxic proteins lack a translocation domain for cytosolic access; therefore, their endosomal escape into the cytosol after endocytosis is inefficient, restricting the potency. These limiting factors may explain the absence of clinical trials of human cytotoxic-proteinebased ITs until now.…”

Small molecules that target the ubiquitin system

“…Rather than inhibiting E3 ligases, targeted protein degradation, including proteolysis targeting chimeras (PROTACs) [73], immunomodulatory drugs (IMiDs) [74], and specific and nongenetic IAP-dependent protein erasers (SNIPER) [75,76], harnesses the destructive power of the host cell ubiquitin machinery to eliminate unwanted disease-causing proteins in a targeted manner. Since a detailed account, this technology is beyond the scope of this review, we refer readers to many recent reviews that comprehensively summarize recent progress in this field [77][78][79][80][81][82][83][84].…”